Composition for attracting blood sucking arthropods and fruit flies

ABSTRACT

The present invention is directed to a composition for attracting blood sucking arthropods and fruit flies. Furthermore, the present invention is directed to a method of attracting blood sucking arthropods and fruit flies and to a kit or trap, comprising the components of said composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the US national phase of PCT applicationPCT/EP03/05980, filed 6 Jun. 2003, published 18 Dec. 2003 WO 03/103395,and claiming the priority of U.S. provisional application 60/386,582filed 7 Jun. 2002.

FIELD OF THE INVENTION

The present invention is directed to a composition for attracting bloodsucking arthropods and/or fruit flies. Furthermore, the presentinvention is directed to a method of attracting blood sucking arthropodsand to a kit or trap, comprising the components of said composition.

STATE OF THE ART

Olfactory cues are widely used by blood sucking insects to detect and tofind sources for blood meals. Since blood sucking arthropods are one ofthe most important groups of vectors for human and animal disease, manyattempts have been undertaken to explore the attractive blend of hostodours. For example, different mosquito species have developed differenthost preferences and it is generally assumed that host selection anddiscrimination is mainly based on olfactory cues. One importantkairomone is CO₂, a major component of breath that has been shown toactivate and to attract a number of blood sucking insect species(Gellees 1980; Takken 1991; Eiras and Japsen 1994; Geier et al. 1999).Another is L-(+)-lactic acid, a component present in human breath aswell as on human skin. This compound is only slightly effective byitself, but acts synergistically with CO₂ and other components fromhuman skin, at least for the Yellow Fever Mosquito Aedes aegypti (Geieret al., 1996). Interestingly, the latter components were found to beattractive only in combination with lactic acid.

Carbon dioxide has been shown to attract mosquitoes. Willis, J. Exp.Zool, 121, 149-179 (1952), discloses that Aedes aegypti are attracted tocarbon dioxide. The role of carbon dioxide in the attraction ofmosquitoes to hosts also has been the subject of numerous laboratorystudies. Rudolfs, N. J. Agric. Exp. Sta. Bull., 367 (1922), and Gouck,J. Econ. Entomol., 55, 386-392 (1962), describe carbon dioxide as anactivator, rather than an actual attractant. However, it is noted thatcarbon dioxide itself, i.e. as a single compound, is less attractive asthe natural host, e.g. human host. Therefore, it may not be serve as apowerful attractant alone.

Acree, et al., Science 161, 1346-7 (1968), disclose that L-lactic acid,isolated from the human hand, attracts female Aedes aegypti. It is alsodisclosed that carbon dioxide is necessary to observe this attraction.

Wensler, Can. J. Zool., 50, 415-420 (1972), discloses the use of ethylether soluble honey odors to attract A. aegypti.

Compositions consisting of lactic acid analogues and carbon dioxide havealso been shown to attract mosquitoes. Carlson, J. Econ. Entomol., 66,329-331 (1973), discloses that some tested analogues of lactic acid hadequivalent attraction to L-lactic acid, but this was not true at alltested doses. The highest reported attraction was 40% of female A.aegypti.

Bar-Zeev et al., J. Med. Entomol., 14, 113-20 (1977), disclose that acomposition consisting solely of lactic acid and carbon dioxide attractsA. aegypti. Here, the lactic acid was dissolved in acetone

Price, J. Chem. Ecol., 5, 383-95 (1979), discloses that human emanationsand carbon dioxide attract female Anopheles quadrimaculatus.

Gillies, Bull. Entomol. Res., 70, 525-32 (1980), reviews the use ofcarbon dioxide to activate and attract mosquitoes. The drawbacks in theuse of carbon dioxide alone as an attractant for blood suckingarthropods are as explained above.

Schreck, J. Chem. Ecol., 8, 429-38 (1981), discloses that materialsisolated from human hands, other than L-lactic acid, attract female A.aegypti and, A. quadrimaculatus mosquitoes.

Lactic acid, in combination with phosphorous-containing compounds havebeen shown to attract mosquitoes. Ikeshoji, Jpn. J. Sanit Zool., 38,333-38 (1987), discloses lactic acid and hempa; lactic acid and metepa;lactic acid, metepa and olive oil; and lactic acid and DDVP attractmosquitoes.

Lactic acid-related compounds have also been tested as mosquitoattractants by electrophysiology. Davis, J. Insect Physiol., 34, 44349(1988), discloses that neurons in the antennae are excited by L-lacticacid, and that analogues of lactic acid, e.g., carboxylic acids,alcohols, hydroxyacids, aldehydes, thiols and haloacids were tested forneuron response. It was shown that no compound elicited as high of arelative responsiveness toward lactic acid-excited cells as did lacticitself. Lactic acid was shown to excite neurons in the antennae ofvirgin A. aegyptiby Davis, J. Insect Physiol., 30, 211-15 (1984). It hasbeen shown that carbon dioxide, in combination with other chemicals,serves as an attractant for mosquitoes. Takken and Kline, J. Am. Mosq.Control Assoc., 5, 311-6 (1989), disclose 1-octen-3-ol (octenol) andcarbon dioxide as mosquito attractants. Van Essen, Med. Vet. Entomol.,63-7 (1993), discloses the use of carbon dioxide, octenol, and light toattract several species of mosquitoes. Takken, J. Insect Behavior, 10,395-407 (1997), discloses that a composition consisting solely of carbondioxide, acetone and octenol attracts several species of mosquitoes.

Kline, Med. Vet. Entomol., 4, 383-91 (1990), discloses that honeyextract, octenol, carbon dioxide, L-lactic acid plus carbon dioxide,L-lactic acid plus octenol plus carbon dioxide attract mosquitoes welland butanone plus carbon dioxide, and phenol alone are less effective.

Schreck, J. Am. Mosq, Control Assoc., 6, 406-10 (1990), discloses thatmaterials isolated from human skin attract female A. aegypti and A.quadrimaculatus, and that the level of attraction, varies from person toperson. It is also disclosed that differences in attraction level arepresent depending on the body location origin of the material.

Takken, Insect Sci. Applic., 12, 287-95 (1991), reviews mosquitoattractants and lists acids, alone or in combination with other aminoacids that are attractive for mosquitoes.

Eiras, Bull. Entomol. Res., 81, 151-60 (1991), discloses that lacticacid, carbon dioxide, human sweat and thermal convection currentsattract female A. aegypti.

Carlson, J. Med. Entomol., 29, 165-70 (1992), discloses that the releaseof carbon dioxide from the human hand is negligible and therefore is nota factor in the attraction of A. aegypti to the human hand.

Bowen, J. Insect Physiol., 40, 611-15 (1994), discloses that lactic acidsensitive receptors are present in Ae. atropalpus.

Eiras, Bull. Entomol. Res., 81, 207-11 (1994), discloses that lacticacid in combination with carbon dioxide has been shown to attractmosquitoes.

Charlwood, Ann. Trop. Med. Parasitol., 89, 327-9 (1995), discloses themosquito-mediated attraction of female mosquitoes to hosts. Severalspecies of mosquitoes were more attracted to a host, e.g., human leg,which already had mosquitoes feeding than a host which had no mosquitoesfeeding on the host (termed “invitation effect”). An apparent pheromone,which was given off by the feeding mosquitoes, was speculated to attractother mosquitoes to the host.

DeJong and Knols, Experientia, 51, 80-4 (1995), discloses that differentmalaria mosquito species (An. gambiae s.s. and An. atroparvus) preferdifferent biting sites on the human body. DeJong and Knols, ActaTropica, 59, 333-5 (1995), disclose that An. gambiae is attracted tocarbon dioxide.

Bernier, Ph.D. Dissertation, University of Florida (1995), discloses thepresence of lactic acid, glycerol, and long chain acids and alcohols onthe skin, as well as other chemicals for a total of over 300 compounds.Some of these were identified and examined as candidate attractants.

Geier, in Olfaction in Mosquito-Host Interactions, 13247 (1996),discloses that carbon dioxide alone is an attractant and that lacticacid alone is a mild attractant, but that the two act as a synergisticattractant. It also discloses that fractions of ethanol washings fromhuman skin are attractive.

McCall, J. Med. Entomol., 33, 177-9 (1996), discloses that A. aegyptiwere attracted to volatile constituents of mouse odor, but did notidentify potential chemicals.

Knols, Bull. Entomol. Res., 87, 151-9 (1997), discloses the use ofLimburger cheese (the acid and non-acid solvent extracted fractions) toattract An. gambiae. Nineteen saturated and unsaturated aliphatic fattyacids, ranging in carbon chain lengths from C2-C18 were identified inLimburger cheese.

Mboera, J. Vector Ecol., 23, 107-13 (1998), disclosed that Culexquinquefasciatus is attracted to a worn stocking and that carbon dioxideplus body odor did not increase response.

Kline, J. Vector. Ecol., 23, 186-94 (1998), disclosed that inolfactometer tests, the human hand or worn sock attracted 80% and 66%,respecively, of A. aegypti in the cage. In comparison, Limburger cheeseattracted 6.4%, and the control 0.0% in the olfactometer.

Bernier, Anal, Chem., 71, 1-7 (1999), discloses the method for analysisof skin emanations, including the identification of lactic acid,glycerol, C12-C18 carboxylic acids and C4-C11 aldehydes.

Geier and Boekh, Ent. Exp. et Appl., 92: 9-19, 1999 describe theresponse of mosquitoes to host odors, e.g. lactic caid and carbondioxide in a small Y-shaped wind tunnel.

Geier et al., Chem. Senses 24: 647-653, 1999, describe lactic acid as anessential synergist for ammonia as an attractant for Aedes aegypti.

Takken and Knots, Annu. Rev. Entomol., 44, 131-57 (1999), reviewedodor-mediated behavior of afrotropical mosquitoes, reaffirming carbondioxide as the best known mosquito kairomone.

Braks and Takken, J. Chem. Ecol., 25, 663-72 (1999), disclose that2-day-old incubated sweat became attractive to An. gambiae.

Geier et al., Chem. Senses 25: 323-330, 2000, describe the attractiveeffect of mixtures containing ammonia, lactic acid plus two fatty acids.

Various chemicals have been disclosed as attractants for mosquitoes.U.S. Pat. No. 4,818,526 to Wilson discloses the use of dimethyldisulfide and dibutyl succinate and combinations thereof as attractantsfor Culicidae (mosquitoes).

U.S. Pat. No. 4,907,366 to Balfour (1990) discloses the use of acomposition consisting solely of lactic acid, carbon dioxide, water, andheat to attract mosquitoes.

PCT WO 98/26661 to Justus discloses mixtures of L-lactic acid and itssodium salt, glycerol, and cheese extracts, with and without unsaturatedlong chain carboxylic acids, alcohols and an amide as attractive for A.aegypti. The glycerol, as well as other components described asequivalent to the glycerol, appear to make the composition substantive,so that it does not evaporate immediately in a rapid pulse. However, theactive ingredients from Limburger cheese, which are the attractantchemicals, are not disclosed within the document, nor were statisticaldata reported for the results used in the examples.

U.S. Pat. No. 6,267,953 B1 to Bernier (2001) discloses the use of acomposition consisting of lactic acid, aceton, and dimethyl disulfid toattract mosquitoes.

Several of the above-mentioned chemicals and chemical compositions havebeen employed to attract any of the hundreds of species of mosquitoesand related arthropods that utilize humans and animals as their hosts.In fact, many of the disclosed compositions have been claimed to beactive as attractants for mosquitoes. However, the activities of theseattractants are often inconsistent and below 50% attraction response inlaboratory experiments. Furthermore, none of the disclosed compositionshave been shown to be able to attract mosquitoes on a consistent basisas efficiently as, or more efficiently than the human body. Moreover,synthetic chemicals that are unsafe for the environment or that may bedangerous to human health have been used in some of the knowncompositions.

Therefore, it is the object of the present invention to provide chemicalcompositions that can be employed safely in the environment, and thatexhibit a synergistic effect for attracting mosquitoes and fruit flieswherein the compositions are as efficient than the human body or moreefficient in attracting mosquitoes and at least as sufficient or moreefficient in attracting fruit flies than fruit.

These objects are solved by the subject-matters of the independentclaims. Preferred embodiments are set forth in the dependent claims.

SUMMARY OF THE INVENTION

The present inventors surprisingly found that a certain mixture ofcomponents is more attractive to blood sucking arthropods and fruitflies than the single components or the same components in binarymixtures. Unexpectedly, it further turned out that a crucial parameterfor attractiveness is the mixing ratio of said components in the gaseousphase. Compositions comprising the same ingredients could be observed asbeing unattractive when leaving a certain range of mixing ratios in thegaseous phase. The mixing ratio in the gaseous phase could also bedefined as that part of the composition, which is directly perceptibleby the arthropods' sense organs.

The term “mixing ratio in gaseous phase” as used herein is to beunderstood as the molar amount and mixing ratio of gaseous components.These components may be provided in liquid, solid, or gaseous form. Whenprovided as liquids or solids, they may be allowed to evaporate in orderto provide the gaseous form of a component. Upon mixing and/ordiffusion, the molar ratio of the compounds relative to each other inthe gaseous phase is defined as “mixing ratio in the gaseous phase”. Theamount of a given compound that is evaporated is termed the “evaporatedamount”. For the purposes of this application, this term shall alsoinclude amounts of compounds provided in the gaseous phase, which havenot evaporated. The amount referred to is the amount of the compound inthe gaseous space where all of the compounds of the inventivecomposition are mixed together.

The compositions disclosed herein are provided to attract blood suckingarthropods and fruit flies. Both “blood sucking arthropod” and “fruitfly” are members of the phylum Arthropoda, which is the largest phylumin the animal kingdom, comprising about 75% of all animals that havebeen described. The estimated number of arthropod species is between1.000.000 and 2.000.000. Arthropods vary in size from the microscopicmites to the giant decapod crustaceans.

The phylum Arthropoda includes many families of insects that are of amedical and veterinary importance, e.g., mosquitoes (Culicidae),blackflies (Simuliidae), sand flies (Phlebotominae), biting midges(Ceratopogonidae), horseflies (Tabanidae), tsetse flies (Glossinidae),stable flies and house flies (Muscidae), fleas (Siphonaptera), lice(Anoplura), triatomine bugs (Triatominae), soft ticks (Argasidae) andhard ticks (Ixodidae).

Preferred blood sucking arthropods include mosquito (Culicidae),blackfly (Simuliidae), sand fly (Phlebotominae), biting midge(Ceratopogonidae), horsefly (Tabanidae), tsetse fly (Glossinidae),stable fly and house fly (Muscidae), flea (Siphonaptera), louse(Anoplura), triatomine bug (Triatominae), soft tick (Argasidae) and hardtick (Ixodidae).

It is appreciated that “mosquito” can be any of the mosquitoes belongingto the suborder diptera known as Nematocera. This suborder includes thefamily Culicidae. The 3400 or so species of mosquitoes are arranged in38 genera. The Culicidae are divided into three subfamilies: theAnophelinae, including the well-known genus Anopheles, many species ofwhich are responsible for the transmission of malaria; theToxorhynchitinae, the large larvae of which eat other mosquito larva;and the Culicinae which, with about 2930 species in about 34 genera, aredivided into two tribes: the Culicini and the Sabethini. The Culcinemosquitoes include such well known genera as Culex, Aedes and Mansonia.The sebethene mosquitoes include Sabethes, Wyeomyia and Malaya. Aspecific mosquito is one belonging to one of the genera Culex, Aedes,Psorophora, Wyeomyia, Mansonia, Coquilletidia and Anopheles.

A preferred blood-sucking arthropod is a mosquito belonging to thegenera Culex, Aedes, Mansonia, Wyeomyia, Psorophora, Coquilletidia orAnopheles. Further preferred blood-sucking arthropods include Simulidae,Triatoninae, Siphonaptera, Tabanidae, Culicoides, Phleobotomines,Muscidae, Glossinidae, Ixodidae or Argasidae.

Further included are, of the subfamily Phlebotominae (sandflies) (i.e.Order: Diptera; Suborder: Nematocera; Infraorder: Psychodomorpha;Superfamily: Psychodoidea; Family: Psychodidae), the Genus: Lutzomyiaand Genus Phlebotomus.

Regarding fruit flies, included are all species of Drosophila.

The composition for attracting blood sucking arthropods and/or fruitflies according to the invention comprises an effective amount of thefollowing:

-   a) at least one compound from group I, III or III or an acceptable    salt thereof or a combination thereof with    -   group I consisting of alpha-hydroxycarboxylic acids,        particularly alpha-hydroxymonocarboxylic acids, each containing        a C₀-C₈ alkyl chain group,    -   group II consisting of alpha-thiomonocarboxylic acids and        alpha-thiodicarboxylic acids, each containing a C₀-C₈ alkyl        chain group,    -   group III consisting of at least one compound of group I or II        wherein the alkyl group is substituted by a C₆-C₁₀ aryl group;-   b) at least one compound which is a C₄-C₈ carboxylic acid or an    acceptable salt thereof, selected from the group consisting of    butyric acid, valeric acid, caproic acid, oenanthic acid, caprylic    acid and variations thereof, wherein said variations are defined as    having one or more unsaturated bonds and/or being branched    carboxylic acids;-   c) ammonia and/or primary amines with C₁-C₆ atoms.

The term primary amine as used herein encompasses all derivatives ofammonia, in which one hydrogen atom has been substituted by an alkylchain of from 1-6 C-atoms.

According to a preferred embodiment, the alkyl chains (group I and/orII) contain 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. It is furtherpreferred that the aryl group of group III is a phenyl group.

Compound a) is preferably selected from glycolic acid, thiolactic acid,lactic acid, thiomalic acid, tartaric acid and/or mandelic acid. It isnoted that the composition according to the present invention is notrestricted to the use of only one of the compounds of group I, II andIII mentioned in feature a), above. It is also comprised by the scope ofthe present invention to use two or more of those components, if theyshow or contribute to synergistic effects as attractants for bloodsucking arthropods or fruit flies.

Instead of caproic acid, heptanoic acid can be preferably used asheptanoic acid is free of antidesired smell. In another embodiment,heptanoic acid is used additionally to caproic acid.

Instead of ammonia, ammonia-releasing compounds can be used. Examplesare (NH₄)₂CO₃ (ammonium carbonate), NH₄HCO₃ (ammonium hydrogencarbonate), (NH₄COONH) (ammonium carbamate, NH₄Cl (ammonium chloride)and other ammonia salts.

Examples for compounds of group I are glycolic acid, lactic acid,tartaric acid.

Examples for compounds of group II are thiolactic acid and thiomalicacid.

An example for a compound of group III is mandelic acid.

Examples for primary amines are ethylamine, propylamine, butylamine andpentylamine.

An example for a branched carboxylic acid of component b) is isovalericacid or isobutyric acid.

According to a preferred aspect, the present invention provides acomposition comprising lactic acid, caproic acid, ammonia, and/oracceptable salts thereof.

As mentioned above, an important parameter for the present compositionsin view of their characteristics as attractant for blood suckingarthropods is the mixing ratio in the gaseous phase.

According to one aspect of the invention, in the composition forattracting arthropods, the components a:b:c are present in a molaramount of about 1:0.1-100:0.01-10 or 1:0.5-50:0.05-5 or 1:1-10:0.1-1with respect to their mixing ratio in gaseous phase.

According to a preferred embodiment of the invention the componentsa:b:c are present in a molar amount of about 1:1:0.6 with respect totheir mixing ratio in gaseous phase.

The compositions for attracting arthropods may further comprise ascomponent d one or more of further blood sucking arthropod attractingcompounds, which preferably are selected from the group of at least oneof C₁-C₃ carboxylic acids and acceptable salts thereof. Those may beselected from the group consisting of formic acid, acetic acid andpropionic acid and at least one of dichlormethane, trichlormethane,acetone, phenol, 1-octen-3-ol, fermenting yeast, and an extract offermenting yeast. An especially preferred component d is acetic acid.

A further preferred composition is one wherein component d comprises atleast one of formic acid, acetic acid, or propionic acid.

The compositions which comprise one or more of components d, preferablycomprise components a:b:c:d in a molar amount of about1:0.1-100:0.01-10:0.01-1000 further preferably about1:0.1-100:0.01-10:0.01-100, more preferably about1:0.1-100:0.01-10:0.01-50 further preferably about 1:1-10:0.1-1:0.1-1more preferably about 1:1-2:0.2-0.8:0.1-0.3 with respect to their mixingratio in gaseous phase.

A preferred composition, comprising components a, b, c and d comprisesan effective amount of lactic acid, ammonia, caproic acid, acetic acidor acceptable salts thereof. The compounds are preferably present in theratios given above, most preferably in a molar amount of about1:1:0.6:0.2 with respect to their mixing ratio in gaseous phase.

According to a preferred embodiment, ammonia is included in a mixingamount of not more than 10 times compared to lactic acid with respect totheir mixing ratio in gaseous phase.

Preferably, the mixing ratio of lactic acid and caproic acid is between10:1 and 1:10 with respect to their mixing ratio in gaseous phase.

If the compositions of the present invention contain ammonia and lacticacid, the mixing ratio of ammonia and lactic acid preferably is between1:1 and 1:50 with respect to their mixing ratio in gaseous phase.

If the compositions of the present invention contain acetic acid andlactic acid, the mixing ratio of acetic acid and lactic acid is between1:1 and 1:100 with respect to their mixing ratio in gaseous phase.

Preferably, the amount of caproic acid is higher than the mixing amountof lactic acid and the amount of ammonia is lower than the amount oflactic acid in the gaseous phase.

It should be emphasized that the compositions of the present inventionare not restricted in quality or quantity to the above mentionedcomponents and their effects, but may additionally contain furtheringredients like stabilizers, fragrances, preservatives, dilutingagents. Also, the compositions may additionally comprise otherattractants for arthropods, such as an effective amount of carbondioxide. Furthermore the effects of the compositions disclosed hereinmay be further enhanced by combining them with other attractants, e.g.visual stimuli, heat, moisture and wind.

The present invention is further directed to traps or kits, whichcontain the above mentioned components. Preferably, those traps or kitscomprise containers or vials, wherein components a, b, c and d (ifpresent) preferably each are located in separated containers or vials.The quantity of the components a, b, c and d is selected to provide aneffective mixing ratio in the gaseous phase (as disclosed above) in arespective environment (dependent on ambient temperature, moisture andthe like).

To effectively control the evaporating amount of components a, b, c andd, the trap or kit may further comprise means for controlled release ofcomponents a, b, c and d.

The compositions of the present invention may be added in any form, to acommercial or home-made trap to enhance the collection of the arthropod.The composition may diffuse out or away from the trap with or without agas stream (e.g., air, carbon dioxide, etc.) as a carrier.

As used herein, a trap is a device that ensnares an arthropod. Effectivetraps include those well known in the art. Suitable traps arecommercially available from different sources (e.g., Mosquito Magnet™,available from American Biophysics, Corp., 2240 South County Trail, EastGreenwich, R.I. 02818-1536, USA., Mosquito Trap MK01 by Lentek, and TheMosquito Trap U.S.A., both available from Comfort House, 189-VFrelinghuysen Ave., Newark, N.J. 07114-1595

The compositions of the present invention may be delivered in vials orother sample containers. The compositions may be blended together in onesingle vial or container, or can be located in separate vials orcontainers, the latter being preferred (see above).

The compositions of the present invention may be delivered in the gasphase, such as by a compressed cylinder. In addition, the compositionexisting in the gas phase, may optionally be mixed or unmixed with aninert carrier gas.

The synergistic attractant compositions of the present invention may beprovided by any number of mechanisms and in different formatsappropriate to particular types of usage. The main function of theformats and mechanisms is to provide release of the attractant over aperiod of time sufficient to attract arthropods (e.g., mosquitoes)effectively, and especially to attract arthropods effectively to anavailable source of arthropod control material (e.g., insecticide,microbial agent, mechanical and electrical killing devices) which iseffective against mosquitoes, and the like, as described above.

The structures used to release the attractant compositions of thepresent invention could be as simple as a tray carrying the composition,a housed tray or other container carrying the compositions, timedrelease canisters or spray cans, absorbent materials retarding therelease of the attractant (e.g., fabric, paper, porous material, foam,absorbent polymer, super absorbent polymer [e.g., the super absorbentacrylic polymers as described in U.S. Pat. No. 5,679,364], containerswith semipermeable membranes, vented containers, glues, and the like).The materials which would more actively attack the arthropods may beassociated with the attractant (in a mixture) or may be located near theattractants so the chemicals do not adversely interact or react.

In addition, combining the compositions of the present invention with aninsecticide provides a means of local extermination, not requiringwide-disbursement of the insecticide. Addition of a slow releasechemical mechanism, such as paraffin, or other suitable viscous chemical(e.g., glycerol), microencapsulation techniques and all controlledrelease devices provide a means to reduce the evaporation rates of thecompositions.

According to a further aspect, the present invention provides a methodof attracting blood sucking arthropods and/fruit flies comprising thestep of exposing the environment with an evaporated composition of anyone of the preceding claims, which composition is effective to attractblood sucking arthropods and/or fruit flies.

The invention also relates to a composition of matter comprising thecomponents a, b, c and preferably d as described above and hereinbelow.The components are preferably present within said composition of matterin a ratio of molar amounts that allow said composition of matter, whenplaced within the means for controlled release of a device of theinvention as described above and hereinbelow, to lead to the release ofa composition of the invention. The means for controlled release arepreferably similar to those described in example 5 and those known inthe art of mosquito traps. Preferably, the molar amounts of thecomponents in the composition of matter correspond to the molar amountsgiven above and hereinbelow for the composition of the invention.Preferred molar amounts are those wherein components a:b:c:d are presentin a molar amount of about 1:0.1-100:0.01-10:0.01-1000. More preferredis a ratio of components a:b:c:d of 1:0.1-100:0.01-10:0.01-100. Furtherpreferred is a ratio of components a:b:c:d of1:0.1-100:0.01-10:0.01-100. More preferred is a ratio of componentsa:b:c:d of 1:0.1-100:0.01-10:0.01-50. Most preferred is a ratio ofcomponents a:b:c:d 1:1-10:0.1-1:0.1-1. All ratio numbers are given withrespect to their mixing ratio in gaseous phase.

A more preferred composition of matter is one is one comprising aneffective amount of lactic acid, ammonia, caproic acid, preferably,acetic acid, and/or acceptable salts thereof. A preferred ratio for thiscomposition is one wherein the components are present in a molar amountas mentioned above. Most preferred is a ratio of about 1:1:0.6:(whenacetic acid is present) 0.2 with respect to their mixing ratio ingaseous phase.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The invention is now illustrated by the following, non-limitativeExamples, Drawings and description thereof. By interchanging thecomponents as identified herein, the skilled artisan will find the bestcomposition to attract either blood sucking arthropods or fruit flies orboth. These variations and the experiments to be made are within theskill of the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the responses of female A. aegypti to differentcombinations of four components of human body odor,

FIG. 2 shows results from a Y-tube test where mosquitoes can choosebetween the scent emanating from a human hand (left side) and the scentresulting from the standard blend as defined in example 1 (right side).

FIG. 3 shows behavioral responses to varying proportions of eachcomponent in the synthetic blend. A, Standard blend and blends that aremodified in the concentration of only one component, B, Directcompetition tests between the standard blend and the modified blends.

FIG. 4 shows the behavioral effect of adding synthetic odor compound tonatural blends (index finger) of humans. A, B and C refer to differentvolunteers.

FIG. 5 shows the olfactometer for testing mosquitoes' odour preference.A, top view, B, arrangement for the production of various concentrationsof odour

EXAMPLE 1

Measurements of attractiveness of different compositions were carriedout using a Y-tube bioassay, similarly to the one described in Dekker etal., Med Vet Entomol. 16, p. 91-98, 2002. FIG. 1 shows the responses offemale A. aegypti to different combinations of four components.Abbreviations of stimuli (dose in μmol/min): LA_(ST)=L-(+)-Lactic acid(0.05); CA_(ST)=Caproic acid (0.03); AM_(ST)=Ammonia (0.09);AC_(ST)=Acetic acid (0.02). Bars (mean±S.E.M.) represent the percentageof mosquitoes trapped in the respective upwind chamber of theolfactometer. Values from 30 trials per treatment were averaged.Treatments were tested in random order; test- and control side switchedin successive tests. For statistical analysis the percentage values ofthe stimulus sides were compared with each other in a one-way ANOVA andLSD post-hoc tests; the letter code above the bars indicate significantdifferences: means with no letter in common are significantly different(P<0.05).

The results presented in FIG. 1 clearly show that a mixture of lacticacid, capronic acid, and ammonia, or the standard blend (lactic acid,capronic acid, ammonia and acetic acid) is similarly attractive for themosquitoes as a human hand placed in front of the tube in the directionof the airstream.

EXAMPLE 2

Whereas in Example 1, the attractant was fed into one tube and the othertube was used as a control, the Y-tube bioassay can also be used inorder to competitively compare two stimuli. In this respect, it is ofinterest to directly compare the compositions of the invention againstthe human body. FIG. 2 illustrates Mosquitoes' choice between the humanhand and the standard blend. The hands of 19 Caucasian test persons weretested in direct competition against the standard blend (Std-blend)consisting of a mixture of LA_(ST), AM_(ST), CA_(ST), and AC_(ST)stimuli (labels according FIG. 1). Bars (means of 8 trials±S.E.M.)represent the percentage of mosquitoes trapped at the respective upwindchamber of the olfactometer. Asterisks indicate significant preferences(P<0.05; t-test for paired samples).

The results presented in FIG. 2 show that despite of variations in therelative attractiveness of the standard blend compared to theattractiveness of a hand of a volunteer in each particular case, theoverall attractiveness of the standard blend compared to humanvolunteers, averaged over about 20 volunteers, is roughly equal or moreattractive than the human body.

Thus it is possible to identify a composition according to theinvention, which is a composition as described above, having anattractiveness equal or better that that of the standard blend, bymeasuring the said composition using a bioassay, such as the Y-tubebioassay described above. The bioassay may be carried out against acontrol, which is devoid of any scent, such as the assay described inexample 1. The attractiveness values are then compared against thoseobtained using e.g., the standard blend or the human body.Alternatively, the bioassay may be carried out as described in example2, where mosquitoes must choose between two alternative flight paths.One of the flight paths would contain the composition to be tested,whereas the other one would contain scent of the human body or ofanother composition, e.g., that of the standard blend. Thus, it ispossible, using e.g., the competitive Y-tube bioassay, to directlycompare the attractiveness of a given composition to the attractivenessof the human body or of another composition, which is preferably thestandard blend described above. The compositions of the invention maytherefore be tested for their activity in various ways. Preferredcompositions are those that are equally attractive or more attractive,compared to the above-described standard blend. Further preferredcompositions of the invention are those that are equally attractive ormore attractive when compared with the body or part thereof of a humanvolunteer. As humans differ in their attractiveness to blood-suckingarthropods, it is preferred that the test be carried out with a numberof volunteers, preferably 5 to 100, more preferably 10 to 50, mostpreferably about 20. The preferred blood-sucking arthropods to be usedfor this test are those that are described above, including mosquito(Culicidae), blackfly (Simuliidae), sand fly (Phlebotominae), bitingmidge (Ceratopogonidae), horsefly (Tabanidae), tsetse fly (Glossinidae),stable fly and house fly (Muscidae), flea (Siphonaptera), louse(Anoplura), triatomine bug (Triatominae), soft tick (Argasidae) and hardtick (Ixodidae). More preferred blood sucking arthropods for use in thetest are mosquitoes belonging to the suborder diptera known asNematocera, including the family Culicidae which includes theAnophelinae, including the well-known genus Anopheles, many species ofwhich are responsible for the transmission of malaria; theToxorhynchitinae, the large larvae of which eat other mosquito larva;and the Culicinae which are divided into two tribes: the Culicini andthe Sabethini. Especially preferred mosquitoes are those that belong tothe genera Culex, Aedes and Mansonia, furthermore Sabethes, Wyeomyia andMalaya. More preferred mosquitoes are those belonging to one of thegenera Culex, Aedes, Psorophora, Wyeomyia, Mansonia, Coquilletidia andAnopheles. Most preferred is A. quadrimaculatus and especially, A.aegypti.

EXAMPLE 3

The effect of varying the proportions of each component in theattractant composition was tested using a bioassay similar to the onedescribed in example 1 and 2. FIG. 3 shows behavioral responses tovarying proportions of each component in the synthetic blend.

A) Standard blend and blends that are modified in the concentration ofonly one component were tested against the control stimulus. Thestimulus labels indicate as to which component and concentration theblend was changed. Numbers of the stimuli labels indicate increasing ordecreasing doses compared to the standard one. For example AM⁻⁵ means afivefold lower dose than AM_(ST), AM₊₃₀ a thirtyfold higher dose thanAM_(ST). Otherwise, experiments were carried out as described for FIG.1., with 20 trials per treatment. For statistical analysis, theresponses to the modified blends were compared with the responses to thestandard blend in a one-way ANOVA and Dunnett LSD post-hoc tests;asterisks above the bars indicate significant (P<0.05) differences tothe standard blend.B) Direct competition tests between the standard blend and the modifiedblends. Bars (means of 20 trials±S.E.M.) represent the percentage ofmosquitoes trapped in the respective upwind chamber. Dark bars representthe standard blend; white bars represent the modified blend (stimuluslabels as in FIG. 3A). Asterisks indicate a significant preference(P<0.05, t-test for paired samples).

The results demonstrate that changes in the concentration of capronicacid, lactic acid and ammonia have a profound effect on theattractiveness of the composition. A five-fold or less increase ordecrease in concentration did not have a significant influence on theattractiveness of the composition, while more extreme changes inconcentration clearly lowered the attractiveness of the composition. Incontrast, an up to 25-fold change in concentration of acetic acid didnot significantly change the attractiveness of the composition.

When the same compositions are measured using direct competitionexperiments (as described in example 2), similar results are obtained(FIG. 3B).

EXAMPLE 4

In this example, the effect of adding certain compounds to the scent ofa finger of a human volunteer was tested, using the set-up as describedfor example 1. The right and left index finger of each test person weretested in direct competition with simultaneous addition of a singlecompound to one finger; Martina (A) is highly, Martin (B) medium, andDoris (C) low attractive. The test components were alternately added tothe right or to the left finger, respectively. Bars (means of 16trials±S.E.M.) represent the percentage of mosquitoes trapped in therespective upwind chamber. Black bars represent the pure finger, whitebars the mixture of finger plus the odor stimulus indicated below thebar; stimulus labels as described in FIG. 3A; asterisks indicate asignificant preference (P<0.05, t-test for paired samples).

The results demonstrate that when adding a certain compound, the scentof the human body of some persons may become more attractive to themosquitoes (e.g. FIG. 4 C, capronic acid in all concentrations). It isequally possible that addition of a compound, especially when used in ahigh concentration, makes the scent less attractive (FIG. 4 A,Ammonia+100). This effect depends upon the person involved. However,addition of acetic acid scent in an up to 50-fold higher concentrationthan that of the standard blend did not change the attractiveness withany of the persons involved.

EXAMPLE 5

The apparatus used for the above-described tests is shown in FIG. 5. TheFigure shows the experimental device, wherein

A) is a top view of the olfactometer for testing mosquitoes' odourpreference. The spatial distribution of odourants in the air areoutlined according to the appearance of TiCl₄ smoke, which was injectedinto the olfactometer instead of the odour stimulus. In the arms of theolfactometer the smoke was homogeneous distributed²⁰. More turbulentodour eddies and filaments emerged in the rectangular chamber, althougha steep gradient between both air streams can still be observed in thejoined straight tube. For each test, 20 female A. aegypti mosquitoeswere used from cultures of Bayer AG in Monheim, 5-15 days old, which hadno blood meal before. Details of the olfactometer and the experimentalprocedure are described elsewhere (Geier, M. & Boeckh, J. Entomol. exp.appl. 92, 9-19 (1999); Geier, M., Bosch, O. J. & Boeckh, J. Chem. Sens.24, 647-653 (1999)).

At stimulus onset, 20 mosquitoes in the start chamber are set free tofly through the olfactometer. During their zig zag flight through thestraight tube they encounter alternately air streams from either arm.Following the favoured air stream during upwind flight the mosquitoesenter the respective upwind chamber where they are counted 30 s afterstimulus onset. The percentage of mosquitoes found in each chamberserves as the measure for attractiveness of the odour stimulus. Odourstimuli were produced by passing purified air through Erlenmeyer flaskswhich contain the odour solution (Geier, M., Bosch, O. J. & Boeckh, J.Chem. Sens. 24, 647-653 (1999); Geier, M., Bosch, O. J. & Boeckh, J. J.exp. Biol. 202, 1639-1648 (1999)). The odour laden air is injected intothe air stream of the olfactometer.

B) shows that by varying the concentration of the compound in theErlenmeyer flask or the flow rate of the passing air a wide range ofodour concentrations can be produced. Table 1 summarises the testedcompounds and how the different stimulus doses were produced. Instead ofsynthetic odourants also a human hand can be placed in the air stream ofthe olfactometer to test the natural blend of skin odourants.

The following table summarises the compounds used in the experimentsdescribed above and gives the concentration used in the differentexperiments. The values listed in the column labelled “concentration”refer to the concentration of the odorant in the erlenmeyer flask thatis part of the device as shown in FIG. 5 and explained in example 5. Theconcentration achieved in the air stream will depend upon the air flow.The air flow rates used are given in the table, however, when adifferently dimensioned apparatus is used, different air flows may beused. The concentration of the compounds in the airstream may easily bemeasured by methods known to the person of skill in the art. They may bederived by calculation from the starting amount of a substance, theamount left in the erlenmeyer flask after a given time, and the airstream that has flowed through the flask and through the main tubeduring said time.

TABLE 1 Tested odourants Flow Stimulus rate^(c) Stimulus dose^(d)Component label^(a) Concentration^(b) Solvent [ml/min] [μmol/min]L-lactic acid LA⁻³⁰ Pure Water 2 0.002 (Geier et. al., 1999a) LA⁻¹⁰ Pure5 0.005 LA⁻⁵ Pure 10 0.01 LA⁻² Pure 20 0.02 LA_(ST) Pure 50 0.05 LA₊₃Pure 150 0.15 LA₊₅ Pure 250 0.25 LA₊₂₀ Pure 1000 1.0 Caproic acid CA⁻³⁰1% Paraffin 3 0.001 (Kafka, 1970) CA⁻⁵ Pure 1 0.006 CA_(St) Pure 5 0.03CA₊₅ Pure 25 0.15 CA₊₂₀ Pure 100 0.6 Ammonia AM⁻³⁰ 0.025% Water 0.30.003 (Geier, 1999b) AM⁻⁵ 0.025% 2 0.02 AM_(St) 0.25% 1 0.09 AM₊₅ 0.25%5 0.45 AM₊₁₀ 0.25% 10 0.9 AM₊₃₀ 0.25% 30 2.65 AM₊₁₀₀ 0.25% 100 9 AM₊₃₀₀0.25% 300 26.5 Acetic acid AC⁻³⁰ 0.0001% Paraffin 3 0.0007 (Kafka, 1970)AC⁻⁵ 0.001% 2 0.004 AC_(St) 0.01% 2 0.02 AC₅ 0.01% 10 0.1 AC₂₅ 0.01% 500.4 AC₅₀ 0.01% 100 0.8 Control Co pure paraffin 300 — (no pure water 300— substance added)

EXAMPLE 6

Herein we investigate if heptanoic acid can replace caproic acid in theattractive mixture.

Olfactometer and Methods

See Example 1, FIG. 5. and table 1.

Results

The behavioural responses of the mosquitoes are summarized in table 2.The responses to the blends containing heptanoic acid did not differsignificantly from the blend with caproic acid and there was nosignificant difference between the two concentrations (Attraction:ANOVA, p=0.257, F=1.382; Activation: ANOVA, p=0.490, F=0.720). Theresults clearly show that heptanoic acid can be used instead ofcaproic-acid in the synthetic mixture.

TABLE 2 Behavioural responses of Ae. aegypti Test chamber Controlchamber Active n Stimulus % T¹ S.E. Stimulus % C² S.E. % A³ S.E 16 Mix81^(b) 3.2* No 0.2 0.3 89^(b) 2.2 16 Mix with 76^(b) 2.9* No 0.1 0.493^(b) 1.4 Oe low 16 Mix with Oe 85^(b) 1.2* No 0.2 0.8 91^(b) 1.9 highMeans from n tests per treatment; each test with 18-22 mosquitoes. ¹Meanpercentage of mosquitoes trapped in the test chamber, ²mean percentageof mosquitoes trapped in the control chamber, ³mean percentage ofmosquitoes which left the release chamber. *significant difference (P <0.01) of mean percentage in test- and control chamber: t-test for pairedsamples. Within all experiments means in the test- or active columnsfollowed by the same letter are not significantly different (P < 0.05,one-way ANOVA: LSD post hoc test). Abbreviations: Mix = lactic acid 1.5ml/min flow rate, ammonia 0.1 ml/min, caproic acid 0.5 ml/min; Mix withOe low = lactic acid 1.5 ml/min, ammonia 0.1 ml/min, heptanoic acid 0.5ml/min; Mix with Oe high = lactic acid 1.5 ml/min, ammonia 0.1 ml/min,heptanoic acid 3 ml/min.

EXAMPLE 7

In this example we show the attractive effect of our blend compositionin laboratory room tests with an ventilator insect trap that sucks inthe flying insects. We also investigated the attractiveness of our blendcompositions to different mosquito species. The results presented intable 3 show that the blend clearly enhances the effectiveness of thetrap. A mixing ratio near to the optimum blend catches is almost asattractive as a human test person. Mixing ratios which are verydifferent to the optimum blend are less attractive. This holds true forall tested species.

TABLE 3 Release and recapture of mosquitoes in a laboratory room usingan insect ventilator trap Mean trapped after 20 min Culex AnophelesAedes Aedes Treatment n quinquefasciatus stephensi aegypti albopictus No6 1.0 0.1 2.3 1.2 attractants Blend A 6 6.7 5.8 8.2 6.4 Blend B 6 2.31.9 1.2 0.3 Blend C 6 3.2 2.6 4.1 2.9 Person K 4  7.0¹ 6.2 9.6 8.3Released 10-14 female mosquitoes per test, 4-14 days old, no blood mealprior to release ¹= number of mosquitoes landed on test person n =number of performed tests Blend A: Mixing ratio of lactic acid, caproicacid and ammonia: 1:2:0.3 Vial 1: evaporation rate of lactic acid: 0.02mmol/h Vial 2: evaporation rate of caproic acid: 0.04 mmol/h Vial 3:evaporation rate of ammonia: 0.006 mmol/h Blend B: Mixing ratio oflactic acid, caproic acid and ammonia: 1:2:80 Vial 1: evaporation rateof lactic acid: 0.02 mmol/h Vial 2: evaporation rate of caproic acid:0.04 mmol/h Vial 3: evaporation rate of ammonia: 1.6 mmol/h Blend C:Mixing ratio of lactic acid, caproic acid and ammonia: 1:200:0.3 Vial 1:evaporation rate of lactic acid: 0.02 mmol/h Vial 2: evaporation rate ofcaproic acid: 4.0 mmol/h Vial 3: evaporation rate of ammonia: 0.006mmol/h

In all experiments the volatiles were applied in different vials filledwith the pure test compounds; each compound was filled in a separatedvial. Using different sizes of the openings of the vials, differentrelease rates could be produced. The released amount of each compoundwas estimated by weight loss data. The compounds were mixed in thegaseous phase using a ventilator.

EXAMPLE 8

In this example we show the data of our field experiments in Brazilusing the same insect trap as in example 7. Using the optimum mixingratio our attractants caught mosquitoes which were present in this areain high numbers compared to a trap without attractants. Surprisingly wealso caught Drosophila sp. in relative high numbers with our optimumblend. This shows that our blend composition is also attractive forDrosophila flies.

Trapping of free flying insects in a domestic environment in BeloHorizonte/Brazil during 5 days.

Traps with different lures Human landing Species No attractants Blend ABlend B rate Drosophila sp. 0.2 31.4 10.4 0 Aedes aegypti 2.0 42.8 13.956.4 Culex sp. 0.7 21.1 7.2 30.3 Plebotomus sp. 0 1.2 2.5 0.8 Trappedinsects/24 h Blend A: Mixing ratio of lactic acid, caproic acid andammonia: 1:2:0.3 Vial 1: evaporation rate of lactic acid: 0.02 mmol/hVial 2: evaporation rate of caproic acid: 0.04 mmol/h Vial 3:evaporation rate of ammonia: 0.006 mmol/h Blend B: Mixing ratio oflactic acid, caproic acid and ammonia 1:520:0.3 Vial 1: evaporation rateof Lactic acid: 0.02 mmol/h Vial 2: evaporation rate of caproic acid:10.4 mmol/h Vial 3: evaporation rate of ammonia: 0.006

The distance between each trap was at least 4 m. Each day the positionof the traps was changed randomly.

In all experiments the volatiles were applied in different vials filledwith the pure test compounds; each compound was filled in a separatedvial. Using different sizes of the openings of the vials, differentrelease rates could be produced. The released amount of each compoundwas estimated by weight loss data. The compounds were mixed in thegaseous phase using a ventilator.

Listing of the dosages of the tested compounds. Stock solutions forpreparing the odour sources were ammonium hydroxide (17093, 25%, Fluka,Buchs), lactic acid (100366, 90%, Merck, Darmstadt), acetic acid (45731,99.8%, Fluka, Buchs), and caproic acid (21529, 99.5%, Fluka, Buchs).

^(a) Stimulus labels refer to the marking of stimuli in figures andtext. Starting from the standard doses labelled with _(ST) the numbersof the stimuli labels indicate increasing or decreasing doses comparedto the standard. For example AM⁻⁵ means a fivefold less dose thanAM_(ST), AM₊₃₀ a thirtyfold higher dose than AM_(ST).

^(b) Concentration (% per vol.) of stimulus solution in Erlenmeyerflask. Solvents were distilled water or paraffin (Uvasol®, 107161,Merck, Darmstadt).

^(c) Flow rate of charcoal filtered air passed through the flask.

^(d) Output of test component injected into the olfactometer ascalculated from calibrations of quoted authors. For acetic acid thedoses were extrapolated from Kafka's (1970) calibration data of butyric-and caproic acid. See also FIG. 1 for description of odour presentation.

1. A composition for attracting mosquitoes, which consists essentiallyof: (a) lactic acid or an acceptable salt thereof; (b) caproic acid oran acceptable salt thereof; and (c) ammonia, in a respective molar ratioof 5:3:9.
 2. A method of attracting mosquitoes at a level ofattractiveness equivalent to a level of attractiveness of a human bodycomprising the step of exposing an environment with an evaporatedcomposition consisting essentially of (a) lactic acid or an acceptablesalt thereof; (b) caproic acid or an acceptable salt thereof; and (c)ammonia, in a respective molar ratio of 5:3:9 which composition iseffective to attract mosquitoes.
 3. The method of attracting mosquitoesdefined in claim 2 further comprising the step of trapping the attractedmosquitoes.
 4. A composition for attracting mosquitoes or fruit flies,which consists essentially of: (a) lactic acid or an acceptable saltthereof; (b) caproic acid or an acceptable salt thereof; and (c)ammonia, in a respective molar ratio of 1:2:0.3.
 5. A method ofattracting mosquitoes or fruit flies comprising the step of exposing anenvironment with the composition defined in claim 4 in an amounteffective to attract mosquitoes or fruit flies.